Studies on Papanicolaou staining. III. Quantitative investigations of orangeophilia and cyanophilia

This paper provides data derived from the visible light absorbance spectra of Papanicolaou stained epithelial cells from the uterine cervix. Twenty-four types of spectra have been considered, namely, those derived from orangeophilic and cyanophilic nuclei and cytoplasms of superficial, intermediate, parabasal and dysplastic cells, and cells of carcinoma in situ and invasive carcinoma. Wavelengths of maximum absorbance and peak absorbances are tabulated. The proportions of bound orange G, eosin Y, aluminum-hematein and light green SF yellowish have been calculated. For the majority of cell types, dyebinding differences between orangeophilic and corresponding cyanophilic substrates were statistically significant. CIE coordinates were calculated from absorbance spectra; again differences between organeophilic and cyanophilic cells were statistically significant in most cases. Although the designation of cells as orangeophilic or cyanophilic is made on the basis of cytoplasmic coloration, the nucleus is also usually orangeophilic or cyanophilic. These nuclear differences are real and not due to the effects of over- and underlying cytoplasm.     

Cyanophilic bodies in cervico-vaginal smears.

Thirty-six out of 171 (21%) cervico-vaginal smears that manifested pronounced squamous epithelial atrophy contained cyanophilic bodies about the size and shape of parabasal cells. These cyanophilic bodies have been misinterpreted as cancer cells. Patients whose smears contained cyanophilic bodies were likely to be elderly, at least ten years postmenopausal, and free of any gynecologic symptoms or abnormalities except those associated with previous surgery. Smears which contained cyanophilic bodies also contained numerous parabasal cells in various stages of degeneration, objects which closely resembled trichomonads, and a heavy background of granular material. A morphologic continuum existed between all of these elements. The conclusion, therefore, is that cyanophilic bodies, spurious trichomonads and the granular material are all derived from degenerating parabasal cells. It is suggested that cyanophilic bodies develop because of the diminished efflux of exfoliated epithelial cells and mucus associated with squamous epithelial atrophy. The ensuing stagnation of parabasal cells allows them to degenerate to an advanced degree. It appears that as some of the parabasal cells degenerative, their nuclear chromatin becomes widely dispersed throughout the cytoplasm, thereby forming cyanophilic bodies.     

Nuclear segmentation of bronchial epithelial cells by minimax and thresholding techniques. A comparison

Two nuclear segmentation methods, Baky's minimax algorithm and thresholding, were compared on a sample of 879 atypical bronchial epithelial cells in sputum. Nuclear-cytoplasmic (N/C) ratios for all cells were determined by each segmentation method and compared to a visually determined value. Cells were categorized by atypia class (from metaplastic through malignant), by staining characteristics (orangeophilic and nonorangeophilic) and by method of digitization (either scanning microphotometry or video system). The method of digitization was confounded by subject differences. The results indicated that with most classes of atypia, N/C ratios determined by minimax were closer to the visually derived values than were those of thresholding, particularly with orangeophilic cells. Both methods become progressively less accurate, as compared to the visual procedure, as the degree of atypia increases.     

Quantitative and qualitative analysis of stain color using RGB computer color specification

OBJECTIVE: To establish standardized Papanicolaou stain for cytology using RGB color specification. This new method was formerly used in DTP software application for computer color specification. STUDY DESIGN: RGB color specification was taken from a color film, optical constituents of which were made into computer software. Cell samples used in this study were from 100 sputum specimens stained with Papanicolaou stain. We analyzed the color tone of the cytoplasm of squamous cells in the smear. RESULTS: The R and B value of eosinophilic cells were demonstrated statistically by different values between normal, borderline atypical and malignant squamous cells. G and B values of light green-philic cells demonstrated a statistical difference between normal, borderline atypical and malignant squamous cells. No significant differences were found in RGB value between normal, borderline atypical and malignant squamous orangeophilic cells. CONCLUSION: Using our own method of analyzing Papanicolaou-stained sputum, a new quantitative and qualitative analysis of stain color for standardized Papanicolaou stain was introduced.     

Immunoperoxidase staining of cervicovaginal smears after radiotherapy.

Cervicovaginal smears from 2 women with postirradiation dysplasia, 4 women with postirradiation squamous cell carcinoma of the cervix, 30 women with irradiation atypia and 5 healthy, nonirradiated women were stained immunohistochemically with six keratin antibodies. For four of the antibodies--CK19 (BA17), EMA, PKK-1 and CAM 5.2--squamous cells showing irradiation atypia, postirradiation dysplasia or postirradiation squamous cell carcinoma were more likely to stain positively than were nonirradiated squamous cells. For three of the antibodies in which multiple squamous cells stained positively, the proportion of squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma staining strongly was equal to or greater than the corresponding overall proportion for squamous cells showing irradiation atypia. This was statistically significant with only one antibody, PKK-1. No statistically significant differences were seen in staining of irradiated and nonirradiated squamous cells by MAK-6 and AE1:AE3. The data show that some keratin antigens are more often expressed in the irradiated groups and that there may be differences in the degree of antigen expression between squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma and squamous cells showing irradiation atypia.     

In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering

Raman micro-spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast-like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non-tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non-tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non-tumour bone cells. A supervised classification model (LDA) was built and showed high cross-validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non-tumour cells, with 96% of the cells being correctly classified either as tumour or non-tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.     

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.     

Microspectrophotometric analysis of intact chromatophores of the Japanese medaka, Oryzias latipes

To investigate the possible photoprotective role of chromatophores in fish, the absorbances of four types of intact chromatophores in adult and larval Japanese medaka were analyzed using microspectrophotometric techniques. The absorbance spectrum of each chromatophore class was obtained from 300 to 550 nm. The absorbance spectra of intact leucophores, melanophores and xanthophores were very similar to the published absorbance spectra of the isolated pure pigments contained in each chromatophore type, pteridines, melanin and carotenoids or pteridines, respectively. Based on these absorbance spectra, leucophores and melanophores should provide the most ultraviolet (UV) photoprotection to fish since the compounds they contain, pteridines and melanin, correspondingly, have strong absorbances in the UV region of the spectrum. Xanthophores containing carotenoids are not likely to provide much protection to fish from UV-induced damage since carotenoids have low absorbances in the UV range. Xanthophores containing colored pteridines, however, may provide somewhat greater UV protection to fish, since pteridines absorb more light than carotenoids in the UV portion of the spectrum. The relative frequency, coverage and thickness of these two types of xanthophores should determine how much protection xanthophores as a chromatophore type would provide against UV-induced damage.     

Oxidative damage to plant DNA in relation to growth conditions.

In this study we investigated the level of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA of Cardamine pratensis plants subjected to different growth conditions trying to answer the question whether factors like light and water accessibility or low temperature may have an impact on the total DNA oxidative damage. The level of this modified nucleoside was determined using HPLC coupled to UV absorbance and electrochemical detection (HPLC-UV-EC). We did not observe any statistically significant differences in 8-oxodG level between DNA of etiolated and light exposed plants as well as between DNA of regularly watered and drought-subjected plants. In contrast, we have shown that chilling (1 degree C for 28 h) brings about the increase of 8-oxodG level in DNA.     

CD of nucleic acids: III. Calculated CD of RNAs from new A, U, G, and C transition-moment parameters

New adenine (A) and uracil (U) π → π* transition-moment parameters have been derived from a recently developed semiempirical procedure. Using conformational energy probabilities based on the Boltzmann equation, the new parameters were assigned by optimizing the calculated CD of cyclic nucleotides against measured CD. The derived A-and U-parameters (along with guanine and cytosine parameters derived previously by the same procedure) have been assessed in CD spectral calculations of some polyribonucleic acid sequences, in assumed A-class geometries. Comparisons have been made between CD spectra calculated from the newly derived parameters and those calculated from parameters obtained from a combination of crystal optical measurements and quantum-mechanical calculations. Although some spectral differences do occur, for the RNA sequences considered, no major disagreements were found in CD spectral signs and shapes, between measurements and calculations. Overall, the results indicate that the newly derived A-, U-, G-, and C-parameters show better agreement between theory and experiment than those used in previous nucleic acid CD calculations.     

Cytopathology of the tall cell variant of thyroid papillary carcinoma.

The tall cell variant of thyroid papillary carcinoma differs from classic papillary carcinoma in its more aggressive clinical behavior, cell type (columnar amphophilic to oxyphilic) and higher frequency of stromal lymphoid infiltrate. A retrospective study of three such cases was made, with an emphasis given to the utility of fine needle aspiration cytology in their identification. Aspirates revealed papillary fronds and cyanophilic and oxyphilic neoplastic cells with a high proportion of nuclear grooves and cytoplasmic inclusions. These nuclear details allowed a specific diagnosis of papillary carcinoma with oxyphil cells as compared to oxyphilic cell follicular tumors. Smears from two cases showed, in addition, lymphoid cells and multinucleate giant cells. In them a diagnosis of coexisting Hashimoto's disease, granulomatous thyroiditis or inflammatory tumor stroma could not be excluded cytologically.     

A MORPHOMETRIC STUDY OF THE ULTRASTRUCTURE OF ECHINOCEREUS ENGELMANNII (CACTACEAE). III. SUBAPICAL AND MATURE TISSUES

A stereological, morphometric study of the ultrastructure of subapical cells, xylem parenchyma, and cortical chlorenchyma of Echinocereus engelmannii shows that each of these cell types has a unique organellar composition. The cells of any of these tissues are unique not only in vacuolation (which is visible at the light microscope level), but also in the nucleus/cytoplasm ratio and the concentrations of mitochondria, chloroplasts, and dictyosomes. Furthermore, the differences between each of these cell types were large and statistically significant. It had previously been found that the cells of each zone of the shoot apical meristems of E. engelmannii are different from those of the other zones, but the present study suggests that, considering the large ranges of structure possible in the nonapical cells of this species, the apical meristem variation should be considered as only a minor difference and that the meristem zones are really quite similar to each other.     

Marker features for malignancy in ectocervical cells. Statistical evaluation

Marker features for malignancy have recently been observed in ectocervical cells, even in cells that are visually normal in appearance. This study assessed the statistical significance of these marker features using a mixed-model nested-design analysis of variance (ANOVA). Features in blue intermediate cells from patients with normal cytology, moderate dysplasia, and severe dysplasia/carcinoma in situ, nonkeratinizing cells from patients with moderate dysplasia, severe dysplasia/carcinoma in situ, and invasive cancer, and dysplastic cells from areas of metaplasia from patients with moderate dysplasia, severe dysplasia/carcinoma in situ, and invasive cancer were tested. ANOVA clearly demonstrated that the marker features differentiate between cells of the same cell type originating from patients in different diagnostic categories. In every instance, the differences owing to the diagnostic category were statistically significantly greater than those caused by patient-to-patient variability. Although the discriminating marker features in the intermediate cells were almost exclusively spectral features reflecting staining differences, morphometric features were also marker features in the dysplastic cells.     

Liquid-based cytology findings of glassy cell carcinoma of the cervix. Report of a case with histologic correlation and molecular analysis

BACKGROUND: Glassy cell carcinoma is a rare form of poorly differentiated carcinoma of the cervix with no obvious squamous or glandular differentiation. Its liquid-based cytology findings have not been described before. CASE: A 46-year-old Filipina presented with vaginal bleeding due to a bulky cervical tumor. The liquid-based cytology preparation was of moderate cellularity and contained small clusters of polygonal to elongated tumor cells admixed with amphophilic, granular, necrotic debris. The malignant cells possessed round to oval nuclei; a thin nuclear membrane; finely dispersed chromatin; prominent, solitary nucleoli; abundant, cyanophilic cytoplasm; and discrete cell borders. Occasional tumor cells showed phagocytosis of polymorphs. The background contained a mixed population of inflammatory cells. Eosinophils, though present, were not readily identified in the cytologic specimen. There was no evidence of dyskeratosis, cytoplasmic vacuolation or koilocytosis. Histologic and ultrastructural examination of the tumor biopsy showed classic features of glassy cell carcinoma. Molecular analysis using polymerase chain reaction and restriction fragment length polymorphism revealed the presence of human papillomavirus (HPV) DNA in the liquid-based cytology sample. The HPV genotype, however, did not belong to any of the commonly encountered prototypes. CONCLUSION: Glassy cell carcinoma of the cervix may show distinct, though subtle, cytomorphologic features in liquid-based preparations. The findings, however, are slightly different from those in conventional cervical smears. Awareness of this rare entity is important, as glassy cell carcinoma is often associated with more aggressive clinical behavior.     

Variability in spectral absorption within cryptophyte phycobiliprotein types

Cryptophytes are known to vary widely in coloration among species. These differences in color arise primarily from the presence of phycobiliprotein accessory pigments. There are nine defined cryptophyte phycobiliprotein (Cr-PBP) types, named for their wavelength of maximal absorbance. Because Cr-PBP type has traditionally been regarded as a categorical trait, there is a paucity of information about how spectral absorption characteristics of Cr-PBPs vary among species. We investigated variability in primary and secondary peak absorbance wavelengths and full width at half max (FWHM) values of spectra of Cr-PBPs extracted from 75 cryptophyte strains (55 species) grown under full spectrum irradiance. We show that there may be substantial differences in spectral shapes within Cr-PBP types, with Cr-Phycoerythrin (Cr-PE) 545 showing the greatest variability with two, possibly three, subtypes, while Cr-PE 566 spectra were the least variable, with only ±1 nm of variance around the mean absorbance maximum of 565 nm. We provide additional criteria for classification in cases where the wavelength of maximum absorbance alone is not definitive. Variations in spectral characteristics among strains containing the same presumed Cr-PBP type may indicate differing chromophore composition and/or the presence of more than one Cr-PBP in a single cryptophyte species.     

Topology and Growth of the Intracytoplasmic Membrane System of Rhodopseudomonas spheroides: Protein, Chlorophyll, and Phospholipid Insertion into Steady-State Anaerobic Cells

An equilibrium density gradient centrifugation study involving the separation of "old" and "new" membranes has been developed to determine the manner in which protein, lipid, and chlorophyll are incorporated into growing intracytoplasmic membranes (chromatophores) of Rhodopseudomonas spheroides. Chromatophores derived from cells grown in an H(2)O-medium had a density of 1.175 to 1.180 g/cm(3) and were readily separable from chromatophores having a density of 1.220 to 1.230 isolated from cells grown in a 70% D(2)O-medium. After a shift from "D(2)O-" to "H(2)O"-based media, only hybrid chromatophores derived from a combination of "heavy" (old) and "light" (new) chromatophore material could be detected. The experimentally determined, median density values for the growing intracytoplasmic membrane system followed a theoretically determined profile which was calculated from the density of full "heavy" and full "light" material assuming random, homogeneous incorporation of new material into old membrane. The distribution of the radioactive labels for protein (leucine) and chlorophyll (delta-aminolevulinic acid) were identical and showed a reproducible displacement of the "old" material to the heavy side of the optical density at 365 nm (OD(365)) absorbance and a displacement of the "new" material to the light side of the OD(365) absorbance profile. Specific phospholipid growth showed no displacement for either the "old" or "new" material from the median absorbance profile.     

Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.

The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.     

The hemoprotein content of Chromatium sp. strain D

1. The hemoprotein content of autotrophic Chromatium strain D has been measured from difference spectra, using solvent-extracted and intact cells.

2. The quantities of the individual cytochromes were estimated by absorbance differences at single wavelengths and by peak-trough differences in spectra obtained from reduced versus oxidized, and CO-treated reduced versus reduced preparations. The concentrations found in intact cells weer 1.15, 0.49 and 0.56 nmoles heme per mg protein for cytochromes c₅₅₃, cc′ and , respectively. In intact cells, the corresponding values were 1.33, 0.47 and 0.67 nmoles heme per mg protein.

3. The mesoheme content of the cells, estimated as pyridine hemochromogen, was 2.08 nmoles heme per mg protein, while the amount of protoheme was negligible.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.